The present invention relates generally to the manipulation of genetic materials and more particularly to the manufacture of specific DNA sequences useful in recombinant procedures to secure the production of analogs of a polypeptide identified as natural thaumatin I which include an aspartic acid residue in the 113th position from the amino terminal end of the polypeptide and may further include a lysine residue at the 46th position.
Thaumatin is an extremely sweet-tasting protein produced in the arils of the fruit of the African shrub Thaumatococcus daniellii Benth. The fruit traditionally has been used in West Africa as a sweetener of palm wine, corn, bread, and sour fruit. Thaumatin, which is about 5,000 times sweeter than sucrose on a weight basis, is produced in at least five forms: thaumatins I, II, a, b and c. These proteins, named in the order of elution from an ion exchange column [Higgenbotham, et al., in Sensory Properties of Foods (Birch, et al., eds.), London: Applied Sciences, pp. 129-149 (1977)], have molecular weights of approximately 22 kilodaltons. Thaumatins I and II are nearly identical proteins, each consisting of a single unmodified polypeptide chain, 207 amino acid residues in length.
Thaumatins I and II are non-toxic proteins, are low-calorie and non-cariogenic, and elicit profound sweet taste responses suggesting a stable interaction between these proteins and human taste receptors. Therefore, thaumatin has potential for use as a sugar substitute, food additive, a sweetness receptor probe and a tool for further elucidation of the taste response.
A plentiful supply of pure thaumatin is required to utilize the protein as a possible food additive and research tool. Because the thaumatin plant requires a tropical climate and insect pollination for successful fruit propagation, there are considerable difficulties involved in greenhouse cultivation of the fruit.
Iyengar disclosed an amino acid sequence for thaumatin I which is shown in Table I below [Iyengar, et al., Eur. J. Biochem., 96, 193-204 (1979)].
TABLE 1 __________________________________________________________________________ ##STR1## ##STR2## ##STR3## ##STR4## ##STR5## ##STR6## ##STR7## ##STR8## ##STR9## ##STR10## ##STR11## ##STR12## ##STR13## ##STR14## ##STR15## __________________________________________________________________________
The amino acid sequence for thaumatin II has been deduced from its nucleotide sequence [Edens, et al., Gene, 18, 1-12 (1982)] and a gene for thaumatin II has been cloned from messenger RNA-derived cDNA. The amino acid sequences of thaumatin I and thaumatin II are very similar and their amino acid sequences differ in only five positions according to the reports of Edens, et al. and Iyengar, et al. The five amino acids in the thaumatin II sequence which differ from those reported by Iyengar, et al. for the natural thaumatin I sequence are the following: lysine instead of asparagine at residue 46; arginine instead of serine at residue 63; arginine instead of lysine at residue 67; glutamine instead of arginine at residue 76; and aspartic acid instead of asparagine at residue 113. Sequence analysis also indicated that thaumatin II is initially translated as a precursor form, preprothaumatin, with both a 22 residue amino-terminal extension and an acidic, six-amino acid carboxy terminal tail. The amino terminal peptide was postulated as a secretion signal based on its hydrophobic character and a compartmentalization role was hypothesized for the carboxy terminal extension.
A great deal of work has been directed toward study of the thaumatin family of polypeptides and much effort has been directed toward the manipulation of genetic materials for the microbial production of thaumatin. The Edens, et al. reference cited above notes that a polypeptide having the native sequence of preprothaumatin II has been microbially produced. More specifically, the reference and European Patent Application Nos. 54,330 and 54,331 disclose cDNA sequences coding for native mature thaumatin II and preprothaumatin II and also disclose cloning vehicles comprising the DNA sequences for use in transformation in microorganisms.
While there exist no reports of the isolation of a gene encoding thaumatin I from natural sources (e.g., by genomic or cDNA cloning) work has also been directed toward microbial synthesis of the thaumatin I polypeptide as identified by Iyengar, et al. In co-owned, pending U.S. patent application Ser. No. 540,634 filed Oct. 11, 1983, the disclosure of which is herein incorporated by reference, techniques for the synthesis of manufactured genes coding for the amino acid sequence of thaumatin I as identified by Iyengar, et al. were disclosed, as were DNA microorganism transformation vectors, fusion genes, transformed microorganisms, the processes for expressing the manufactured gene and for securing the polypeptide product produced thereby. Specific manufactured genes of the application incorporated a number of codons "preferred" for expression in yeast host cells.
In U.S. patent application Ser. No. 540,634, procedures are also disclosed whereby the thaumatin II coding sequence could be constructed by primer-directed mutagenesis and fragment excision/religation techniques to a manufactured gene encoding the Iyengar, et al. thaumatin I sequence. Three amino acid changes (Asn.sup.46 to Lys.sup.46, Ser.sup.63 to Arg.sup.63 and Lys.sup.67 to Arg.sup.67) could be accomplished in a single mutagenesis procedure with two primers. The conversion from arginine to glutamine at residue number 76 could be performed in a separate mutagenesis procedure. Still another mutagenesis procedure would be required to effect the change from asparagine to aspartic acid at residue number 113. Alternatively, the changes in amino acid residues 46, 63, 67 and 76 could be effected by synthesis of four oligonucleotide fragments which would be duplexed, polymerized and digested to replace the KpnI and EcoRI duplex of the sequence in pING301. The change at residue 113 would nevertheless require an in vitro mutagenesis procedure. U.S. patent application Ser. No. 540,634 also suggests the manufacture of thaumatin analogs.